PAUL W. CZOTY - Primate Testing - 2007

Abstract: DESCRIPTION (provided by applicant): Although the
subjective effects of cocaine are understood to play an important role
in cocaine abuse, studies in humans have revealed an incomplete overlap
between discriminative stimulus (SD) and reinforcing (SR) effects of
drugs. Moreover, the lack of a clear understanding of the precise roles
of dopamine (DA), serotonin (5-HT) and norepinepherine (NE) in these
effects has hindered efforts to develop medications for cocaine
dependence. The overarching goals of the research in this proposal are
to gain a better understanding of the relationship between the SD and SR
effects of cocaine and to better elucidate the pharmacological and
neurochemical mechanisms that underlie these effects. To accomplish
these aims, rhesus monkeys will be trained to discriminate a
response-contingent injection of 0.1 mg/kg cocaine from saline, with an
opportunity to self-administer 0.1 mg/kg cocaine under a second-order
schedule immediately following the discrimination component. Using this
procedure, the effects of a range of doses of cocaine, other indirect
and direct DA receptor agonists will be characterized, including direct
agonists that differ in efficacy at stimulating D1- and D2-like DA
receptors (Specific Aim 1). To characterize the extent of overlap of the
neurochemical mechanisms involved in production of these abuse-related
effects of cocaine, parallel microdialysis studies will measure
extracellular DA in the ventral striatum during discrimination and self-
administration components of selected doses (Specific Aim 2). Mechanisms
by which 5-HT and NE can modulate the behavioral effects of cocaine will
be examined in subsequent behavioral and microdialysis studies (Specific
Aim 3) that characterize the effects of 5-HT and NE indirect and direct
agonists on the SD, SR and neurochemical effects of cocaine. By
assessing behavioral and neurochemical effects within a behavioral
session in the same monkeys, these innovative studies will: (1) better
describe the importance of SD effects to self-administration, (2) more
clearly elucidate dopaminergic mechanisms involved in production of the
abuse-related effects of cocaine, and (3) provide a unique
characterization of pharmacological and neurochemical mechanisms that
will aid the development of effective pharmacotherapies for cocaine
dependence. Relevance: The proposed studies will provide unique
information about the neurobiological mechanisms through which the
addictive effects of cocaine are produced. Importantly, the results will
provide novel information to aid efforts to develop effective
medications for cocaine addiction, and will enhance our understanding
and interpretation of data collected in animal models of drug addiction.

Public Health Relevance: This Public Health Relevance is not available.

Subjects
Thirteen adult male cynomolgus monkeys (Macaca fascicularis) served as
subjects. Five of the monkeys were drug and experimentally-naïve at the
outset of these studies and lived individually in stainless steel cages.
The remaining 8 monkeys had extensive histories of cocaine
self-administration and exposure to D1 agonists (Czoty et al. 2004,
2005), and were socially housed. Although monkeys occupied both dominant
and subordinate ranks in the social hierarchy, no influence of social
rank was observed in this experiment; thus, data are collapsed across
ranks. All monkeys were fitted with aluminum collars (Primate Products,
Redwood City, Calif., USA) and trained to approach the front of the cage
to be guided into a restraint chair (Primate Products) using a specially
designed stainless steel pole (Primate Products). Monkeys' were weighed
weekly and fed enough food daily (Purina Primate Chow and fresh fruit)
to maintain at least 95% of free-feeding weight; water was continuously
available in the home cage. All procedures were performed in accordance
with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals and were approved by the Animal Care and Use
Committee of Wake Forest University. Environmental enrichment was
provided as outlined in the Animal Care and Use Committee of Wake Forest
University Nonhuman Primate Environmental Enrichment Plan.

Apparatus
The apparatus consisted of a ventilated, sound-attenuating experimental
chamber (1.5 × 0.74 × 0.76 m; Med Associates, East Fairfield, VT). Two
retractable response levers (5 cm wide) were located on one side of the
chamber with a horizontal row of three stimulus lights 14 cm above each
lever and a food receptacle located between the levers. The receptacle
was connected with a Tygon tube to a pellet dispenser (Gerbrands Corp.,
Arlington, MA) located on the top of the chamber for delivery of 1-g
banana-flavored food pellets (Bio-Serv, Frenchtown, NJ). An infusion
pump (Cole-Parmer, Inc., Chicago, IL) was located on the top of the
chamber. Experimental sessions were conducted 5-7 days/week while
monkeys were seated in a primate restraint chair within the operant
conditioning chamber.

Catheter Implantation
All subjects were surgically prepared with a chronic indwelling venous
catheter into a major vein (femoral, internal or external jugular or
brachial) and subcutaneous vascular access port (Access Technologies,
Skokie, Ill., USA) under sterile conditions as previously described (Czoty
et al. 2004). To prolong patency, each port and catheter was flushed
with a solution of heparinized saline (100 U/ml) each day.

ProcedureExperiment 1: Effects of noncontingent cocaine on response
allocation during concurrent availability of saline and food. Subjects
(n=8) in this experiment had extensive experience self-administering
cocaine under a concurrent FR 50 schedule of cocaine and food
presentation described previously (Czoty et al. 2005). Briefly, 50
consecutive responses on one lever produced an injection of cocaine;
completion of an FR 50 on the other lever produced a food pellet.
Responses emitted on the alternate lever before completion of an FR 50
reset the response requirement. Sessions ended after 30 total
reinforcers had been earned or 60 min had elapsed, whichever came first.
Under this schedule, response-contingent cocaine (0.003-0.1 mg/kg)
presentation resulted in dose-dependent increases in the percentage of
responses emitted on the injection lever (Czoty et al. 2005). In the
present studies, saline was substituted for a dose of cocaine that
resulted in >90% injection-lever responding (0.03 or 0.1 mg/kg per
injection). When response allocation stabilized, such that < 5% of the
total responses were allocated to the injection lever for three
consecutive days, a noncontingent injection of cocaine (0.01-0.56 mg/kg,
i.v.) was administered immediately before the session. Each dose was
administered in mixed order across monkeys and the entire cocaine
dose-response curve was determined under the saline, food choice
conditions before re-establishing cocaine self-administration.

Experiment 2: Effects of cocaine self-administration on
reinstatement of extinguished food-reinforced responding. Cocaine-naïve
subjects (n=5) were trained to respond under an FR 50 schedule of food
presentation on the right lever. Sessions began with illumination of the
white light above the lever; completion of 50 responses resulted in the
white light being extinguished, illumination of the red light for 10 sec
and delivery of a food pellet, followed by a 10-sec timeout period in
which the chamber was dark and responding had no scheduled consequences.
The left lever was not extended into the chamber. Sessions lasted until
30 reinforcers had been obtained or 60 min had elapsed. Once response
rates and number of reinforcers were deemed stable, the food pellet
dispenser was unplugged and food-maintained responding was extinguished.
Under these conditions, 50 responses still produced changes in stimulus
lights but no food pellet was delivered. Responding was deemed
extinguished when the number of reinforcers earned was less than 20% of
those earned during baseline sessions for three consecutive sessions.
Next, food pellets (1 or 5) or cocaine injections (0.03-1.0 mg/kg, IV)
were administered non-contingently immediately prior to the session. It
is important to note that, because catheters were flushed with
heparinized saline immediately before each extinction session, data from
non-cocaine priming sessions represent the effects of a priming
injection of saline. Food pellets were delivered into the food
receptacle via manual operation of the pellet dispenser. After a food or
cocaine prime was examined during an extinction session, at least one
baseline extinction session was conducted before the next prime was
administered. The order of presentation of these stimuli was randomized
across subjects.

When the effects of noncontingent cocaine and food had been
determined, monkeys were exposed to cocaine self-administration on the
opposite (left) lever in the presence of the white light; the right
lever remained retracted during cocaine self-administration sessions.
Cocaine- and food-reinforced responding were maintained on opposite
levers so that the contingencies for the different reinforcers would be
associated with topographically different responses. With the white
light illuminated, responding was maintained under an FR 50 schedule of
0.03 mg/kg per injection cocaine for 11 ± 0.6 sessions (range: 10-13
sessions). The dose was chosen based on previous results within our
laboratory that this is the lowest dose that maintains responding above
saline (control) levels (Czoty et al., 2005) and the duration of
self-administration was chosen to conservatively assure that acquisition
of cocaine self-administration had occurred, which we previously
established to be within 7 sessions (e.g., Nader et al., 2002). At the
end of that period, food-reinforced responding was re-established on the
original (right) lever while the left lever remained retracted. When
food-maintained responding was stable, responding was extinguished and
the effects of noncontingent food and cocaine were re-determined as
described above.

Please email: PAUL W. CZOTY,
pczoty@wfubmc.edu to protest the inhumane use of animals in this
experiment. We would also love to know about your efforts with this
cause:
saen@saenonline.org

Rats, mice, birds, amphibians and other animals have
been excluded from coverage by the Animal Welfare Act. Therefore research
facility reports do not include these animals. As a result of this
situation, a blank report, or one with few animals listed, does not mean
that a facility has not performed experiments on non-reportable animals. A
blank form does mean that the facility in question has not used covered
animals (primates, dogs, cats, rabbits, guinea pigs, hamsters, pigs,
sheep, goats, etc.). Rats and mice alone are believed to comprise over 90%
of the animals used in experimentation. Therefore the majority of animals
used at research facilities are not even counted.